Small Peptides And Methods For Treatment Of Chronic Inflammatory Bowel Disease

ABSTRACT

Methods for treating cutaneous inflammation, are described. Also described is a method for inhibiting the mucous release into airways of a patient, a method for blocking IgE activation of a lymphocyte, a method for stabilizing the cell membrane of a lymphocyte, thereby preventing their further involvement in the increased inflammatory response to an IgE antigen challenge, and a method for inhibiting the migration of T-cells. Such methods involve administering to said patient a therapeutically effective amount of a peptide having the formula f-Met-Leu-X, wherein X is selected from the group consisting of Tyr, Try-Phe, Phe-Phe and Phe-Tyr.

RELATED APPLICATIONS

This application is a Division of U.S. application Ser. No. 10/147,633filed May 16, 2002, which is a Division of U.S. application Ser. No.09/190,043 filed Nov. 10, 1998 and issued as U.S. Pat. No. 6,391,856which claims priority from Provisional Application Ser. No. 60/065,336filed Nov. 13, 1997.

FIELD OF THE INVENTION

This invention relates to small peptides having mast cell degranulationinhibition activity and to methods for treating inflammation, andparticularly to N-formyl-methionyl peptides useful for the treatment ofallergies such as allergic rhinitis, uticaria, anaphylaxis, drugsensitivity, food sensitivity, and the like, cutaneous inflammation suchas dermatitis, eczema, psoraisis, contact dermatitis, sunburn, aging,and the like, and arthritis such as osteoarthritis, psoriatic arthritis,lupus, spondylarthritis, and the like. These peptides also are usefulfor treating chronic obstruction pulmonary disease and chronicinflammatory bowel disease. More particularly, the peptides can be usedto replace corticosteroids in any application in which corticosteroidsare used including immunosuppression in transplants and cancer therapy.

BACKGROUND OF THE INVENTION

Asthma is a complex disorder. Both hereditary and environmentalfactors—allergies, viral infections, irritants—are involved in the onsetof asthma and in its inflammatory exacerbations. More than half ofasthmatics (adults and children) have allergies; indeed, allergy tohouse dust mite feces is a major factor in the development of thedisease and in the occurrence of exacerbations. Infection withrespiratory syncytial virus during infancy is also highly associatedwith the development of asthma, and viral respiratory infections oftentrigger acute episodes.

The introduction three decades ago of bronchodilatingβ₂-agonists—adrenergic agonists selective for the β₂receptor—revolutionized the treatment of asthma. These agents proved tobe more potent and longer acting (4-6 hours) than the nonselectiveadrenergic receptor agonists such as isoproterenol, which stimulate bothα- and β-adrenergic receptors. β₂-agonists give rapid symptomatic reliefand also protect against acute bronchoconstriction caused by stimilisuch as exercise or the inhalation of frigid air. Frequency of use canalso serve as an indicator of asthma control. Recently, an extralong-acting β₂-agonist-salmeterol (duration up to 12 hours) wasintroduced in the United States. Salmeterol is so potent that it maymask inflammatory signs; therefore, it should be used with ananti-inflammatory.

Theophylline is a relatively weak bronchodilator with a narrowtherapeutic margin (blood level monitoring is recommended to avoidtoxicity) and a propensity for drug interactions (competition forhepatic cytochrome P450 drug-metabolizing enzymes alters plasma levelsof several important drugs metabolized by that same system).

Moderate asthma is treated with a daily inhaledanti-inflammatory-corticosteroid or mast cell inhibitor (cromolyn sodiumor nedocromil) plus an inhaled β₂-agonist as needed (3-4 times per day)to relieve breakthrough symptoms or allergen- or exercise-inducedasthma. Cromolyn sodium and nedocromil block bronchospasm andinflammation, but are usually effective only for asthma that isassociated with allergens or exercise and then, typically, only forjuvenile asthmatics. Inhaled corticosteroids improve inflammation,airways hyperreactivity, and obstruction, and reduce the number of acuteexacerbations. However, it takes a month before effects are apparent andup to a year for marked improvement to occur. The most frequent sideeffects are hoarseness and oral candidiasis. More serious side effectshave been reported—partial adrenal suppression, growth inhibition, andreduced bone formation—but only with the use of higher doses.Beclomethasone, triamcinolone, and flunisolide probably have a similarmg-for-mg potency; the newer approvals budesonide and fluticasone aremore potent and reportedly have fewer systemic side effects.

Even patients with mild disease show airways inflammation, includinginfiltration of the mucosa and epithelium with activated T cells, mastcells, and eosinophils. T cells and mast cells release cytokines thatpromote eosinophil growth and maturation and the production of IgEantibodies, and these, in turn, increase microvascular permeability,disrupt the epithelium, and stimulate neural reflexes andmucus-secreting glands. The result is airways hyperreactivity,bronchoconstriction, and hypersecretion, manifested by wheezing,coughing, and dyspnea.

Traditionally, asthma has been treated with oral and inhaledbronchodilators. These agents help the symptoms of asthma, but donothing for the underlying inflammation. Recognition during the last 10years of the importance of inflammation in the etiology of asthma hasled to the increased use of corticosteroids, but many patients continueto suffer from uncontrolled asthma.

Scientists have determined that the leukotrienes (of which there are A,B, C, D, and E subtypes) plays a crucial role in asthma. They causeairways smooth muscle spasm, increased vascular permeability, edema,enhanced mucus production, reduced mucociliary transport, and leukocytechemotaxis.

Like related prostaglandin compounds, leukotrienes are synthesized fromarachidonic acid in the cell membrane. Arachidonic acid in mast cells,eosinophils, macrophages, monocytes, and basophils is formed frommembrane phospholipids by the activation of phospholipase A2. After itsformation, arachidonic acid undergoes metabolism via two major pathways:the cyclooxygenase pathway (which produced various prostaglandins andthromboxanes) and the 5-lipoxygenase pathway (which producesleukotrienes). A schematic of arachidonic acid metabolism is illustratedin FIG. 4. The prostaglandins, thromboxanes, and leukotrienes are knowncollectively as eicosanoids.

Anti-leukotrienes are members of a heterogeneous class of anti-asthmaagents with the potential to interfere with the initial steps in theinflammatory cascade. Leukotrienes are inflammatory substances relatedto prostaglandins; both are generated from arachidonic acid in cellmembranes. After arachidonic acid in mast cells, eosinophils,macrophages, monocytes, and basophils is formed, it is metabolized viatwo major pathways: (1) a cycloxygenase pathway (which producesprostaglandins and thromboxanes) and (2) the 5-lipoxygenase pathway,which produces leukotrienes in the cytoplasma. The leukotrienes are wellknown in medical science as the slow reacting substance of anaphylaxis(“SRS-A”). Leukotrienes play an important role in bronchialinflammation. They induce migration, adhesion and aggregation of variouswhite blood cells (e.g., neutrophils, eosinophils, and monocytes) toblood vessels, increase capillary permeability, and cause bronchial andvessel smooth muscle constriction. The results include interstitialedema, leukocyte chemotaxis, mucus production, mucociliary dysfunction,and bronchospasm in the lungs. Certain classes of leukotrienes, forexample, the cysteinyl leukotrienes (LTD₄), are particularly potentbronchoconstrictors, being approximately 100 to 1,000 times more activethan histamine. Leukotrienes, including cysteinyl leukotrienes, arereleased from mast cells during degranulation.

A number of anti-leukotrienes that either block leukotriene receptors orprevent leukotriene synthesis by blocking the enzyme 5-lipoxygenase areunder investigation and in commercial use. The leukotriene inhibitorsare heterogeneous in action: some block 5-lipoxygenase directly, someinhibit the protein activating 5-lipoxygenase, and some displacearachidonate from its binding site on the protein. The leukotrieneantagonists, by contrast, block the receptors themselves that mediateairways hyperactivity, bronchoconstriction, and hypersecretion.

Human lung mast cells produce tumor necrosis factor (TNF), IL-4 and IL-5after IgE stimulation in vitro (Chest 1997; 1 12:523-29).Immunohistochemical analysis in endobronchial biopsy specimens hasconfirmed this together with IL-6 production. Further, mast cell countsand TNF are statistically more significant in asthmatics when comparedto normal subjects. TNF and IL-4 can potentiate up-regulation of theexpression of vascular cell adhesion molecule-1 (VCAM-1)—an adhesionmolecule of the immunoglobin super family—in the endothelial layer ofthe bronchial vasculature. Eosinophils, basophils and mononuclear cellsdisplay the very late activation antigen 4 (VLA-4) integrin on theircellular surfaces, which interacts with VCAM-1. Thus, through theinteraction VLA-4/VCAM-1, TNF and IL-4 facilitate the recruitment ofcirculating leukocytes. The capacity of mast cells to release preformedcytokines (TNF) on IgE-mediated stimulus or to rapidly synthesize others(IL-4, IL-5) could be the initial event leading to bronchialinflammation. In fact, the induction and activation of TH₂ clones,through a further production of cytokines, facilitates the activationand recruitment of the eosinophils, which act as the terminal effectorsof the inflammatory reaction. In turn, the cytokines produced byleukocytes (TH₂ cells, in particular) profoundly affect the development,activation, and priming of mucosal mast cells, thus promoting a positiveproinflammatory loop. The recent findings that human mast cells produceIL-8 and that murine pulmonary-derived mast cells express bothchemokines, monocyte chemoattractant protein-1 and macrophageinflammatory protein-1. This suggests that, besides the cytokinesclassically involved in leukocyte recruitment (IL-4, IL-5, TNF), mastcells also elaborate additional, potent chemoattractants in the airways,acting on eosinophils and polymorphonuclear leukocytes (IL-8). Moreover,because chemokines acting as histamine-releasing factors elicit mastcell degranulation, they may further sustain an autocrine activatingloop.

The mast cells also play a key role in B-cell growth to provide the cellcontact (like basophils) that is required, along with IL-4, for IgEsynthesis in vitro, which suggests that mast cells may directly regulatethe production of IgE independently of T-cells, and may, upon IgEcross-linking, generate a sufficient amount of IL-4 to initiate a localTH₂ response, the subset of T-cells considered to play a central role inatopic asthma. Moreover, mast cells can also act as anantigen-presenting cell to T-lymphocytes, suggesting an even larger rolefor mast cells in the immune network of asthma.

Inhibition of mast cell degranulation byN-formyl-methionyl-leucyl—phenylalanine was reported in Inflammation,Vol. 5, No. 1, pp. 13-16 (1981). There, it was reported that twostructurally different chemotactic peptides, i.e., pepstatin andN-formyl-methionyl-leucyl-phenylalanine, inhibit the increase invascular permeability produced by intradermal injection of 40/80,anti-rat IgE serum, or macromolecular anionic permeability factorisolated from calf lung in rat skin. It also has been reported thatthese peptides appear to act directly on the mast cells.

Because of the importance of treating inflammatory diseases in humans,particularly, for example, asthma, arthritis and anaphylaxis, newbioactive compounds having fewer side effects are continually beingsought. The inhibition of mast cell degranulation by the intervention ofnovel peptides of the present invention within the context of the asthmainflammatory process is visually depicted in FIG. 4.

SUMMARY OF THE INVENTION

The present invention provides methods for treating a variety ofindications using novel pharmaceutical compositions containing in asuitable pharmacological carrier a N-formyl-methionyl-leucyl(“f-Met-Leu”) peptide having mast cell degranulation inhibitionactivity. Particularly useful are those peptides having the formulaf-Met-Leu-X where X is selected from the group consisting of Tyr,Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID.No. 3). Such peptides are useful for treating allergies such as allergicrhinitis, uticaria, anaphylaxis, drug sensitivity, food sensitivity,etc. and the like, cutaneous inflammation such as dermatitis, eczema,psoraisis, contact dermatitis, sunburn, aging, etc. and the like, andarthritis such as osteoarthritis, psoriatic arthritis, lupus,spondylarthritis, etc. and the like. These peptides also are useful fortreating chronic obstruction pulmonary disease and chronic inflammatorybowel disease. The peptides of the present invention can be used toreplace corticosteroids in any application in which corticosteroids areused including immunosuppression in transplants and cancer therapy.

In accord with the present invention, a method for treating an allergyreaction in a mammal comprises administering to the mammal ananti-allergic effective amount of a peptide having the formulaf-Met-Leu-X where X is selected from the group consisting of Tyr,Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID.No. 3). For treating allergies connected e.g. with nasal membranes, apreferred mode of administration is by inhalation. For treating contactallergies, a preferred mode of administration is topical applicationusing a suitable pharmacological carrier. Intradermal injection ortablets can be used for systemic treatments.

The present invention also provides a method for treating cutaneousinflammation in a mammal. The method comprises administering to themammal an anti-inflammatory effective amount of a peptide having theformula f-Met-Leu-X where X is selected from the group consisting ofTyr, Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQID. No. 3).

The present invention further provides a method for treating arthritisselected from the group consisting of osteoarthritis, psoriaticarthritis, lupus, spondylarthritis, and the like in a mammal. The methodcomprises administering to the mammal an anti-arthritic effective amountof a peptide having the formula f-Met-Leu-X where X is selected from thegroup consisting of Tyr, Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No.2) and Phe-Tyr (SEQ ID. No. 3).

In accord with another embodiment, the invention provides a method forinhibiting the infiltration of eosinophils into airways of a patient.The method comprises administering to said patient a airway eosinophilinfiltration inhibiting effective amount of a peptide having the formulaf-Met-Leu-X, wherein X is selected from the group consisting of Tyr,Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID.No. 3).

In accord with a further embodiment, the invention provides a method forinhibiting mucous release in airways in a patient. The method comprisesadministering to the patient an airway mucous release inhibitingeffective amount of a peptide having the formula f-Met-Leu-X where X isselected from the group consisting of Tyr, Tyr-Phe (SEQ ID. No. 1),Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID. No. 3).

Also, the invention provides a method for treating chronic obstructionpulmonary disease in a patient. The method comprises administering tothe patient a chronic obstruction pulmonary disease treatment effectiveamount of a peptide having the formula f-Met-Leu-X where X is selectedfrom the group consisting of Tyr, Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQID. No. 2) and Phe-Tyr (SEQ ID. No. 3).

In addition, the present invention provides a method for treatingchronic inflammatory bowel disease in a patient. The method comprisesadministering to the patient a chronic inflammatory bowel diseasetreatment effective amount of a peptide having the formula f-Met-Leu-Xwhere X is selected from the group consisting of Tyr, Tyr-Phe (SEQ ID.No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID. No. 3).

The invention further provides a method for blocking IgE activation oflymphocytes such as, for example, macrophages, monocytes, eosinophils,neutrophils, TNF and the like. The method comprises contacting saidlymphocytes with an IgE activation blocking effective amount of apeptide having the formula f-Met-Leu-X where X is selected from thegroup consisting of Tyr, Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No.2) and Phe-Tyr (SEQ ID. No. 3).

The invention also provides a method for stabilizing the cell membraneof lymphocytes such as, for example, macrophages, monocytes,eosinophils, neutrophils, TNF and the like, thereby preventing theirfurther involvement in the increased inflammatory response to an IgEantigen challenge. The method comprises contacting said lymphocytes witha cell stabilization effective amount of a peptide having the formulaf-Met-Leu-X where X is selected from the group consisting of Tyr,Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQ ID.No. 3).

Further, the invention also provides a method for inhibiting themigration of T-cells such as, for instance, CD4+ cells, therebypreventing their involvement in the production of IL-4 and IL-5, as wellas other chemokines. The method comprises contacting said T-cells with aT-cell migration inhibiting effective amount of a peptide having theformula f-Met-Leu-X where X is selected from the group consisting ofTyr, Tyr-Phe (SEQ ID. No. 1), Phe-Phe (SEQ ID. No. 2) and Phe-Tyr (SEQID. No. 3).

In certain preferred embodiments of the present invention, patients canbenefit by administering the peptide of the present invention incombination with a second active ingredient. Particularly useful otheractive ingredients for such combination in accord with the presentinvention are, for example, antileukotrienes, β₂ agonists,corticosteroids, and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a log dose response curve illustrating area of capillarypermeability for various concentrations of Compound 48/80.

FIG. 2 is a dose response curve for inhibition of capillary permeabilityby various concentrations of f-Met-Leu-Phe.

FIG. 3 is a dose response curve for inhibition of capillary permeabilityby various concentrations of a preferred peptide of the presentinvention.

FIG. 4 is a schematic illustration of the major pathways for arachidonicacid metabolism further illustrating inhibition of mast celldegranulation.

FIGS. 5A and -5B are schematic illustrations of the different protocolsof Standard (5A) and Resolution (5B) used in The OVA-induced BronchialAsthma Mouse Model.

FIGS. 6A-6D are micrographs illustrating the comparative histopathologyof a treatment with a compound of the present invention inhibiting theOVA induced asthma in treated mice and control mice.

FIG. 7 is a histogram showing the results for treatment in accord withthe present invention on formation of mucus plugs in a murine asthmamodel.

FIGS. 8A-8C show the histopathology of lung tissues of mice treated inaccord with the present invention after induced with asthma.

FIGS. 9A-9D show the histopathology of lung tissues of a second group ofmice treated in accord with the present invention after induced withasthma.

FIGS. 1A-10D show the histopathology of lung tissues of a third group ofmice treated in accord with the present invention after induced withasthma.

FIG. 11 is a graph illustrating the distribution of inflammatory cellsin the aveoli of lungs recovered from OVA-induced asthmatic mice

FIG. 12 is a graph illustrating the airway plug score in airways ofOVA-induced asthmatic mice.

FIG. 13 is a graph illustrating the white cell migration in airwaysOVA-induced asthmatic mice.

FIG. 14 is a graph illustrating the total cells recovered from lunglavage of OVA-induced asthmatic mice.

DETAILED DESCRIPTION OF THE INVENTION

In accord with the present invention, certain small peptides having theformula f-Met-Leu-X where X is selected from the group consisting ofTyr, Tyr-Phe, Phe-Phe and Phe-Tyr have been found to have surprisingactivity for inhibiting the degranulation of mast cells. As a result,such peptides inhibit the release of cytokines (such as, for example,TNF), as well as histamines and leukotrienes and they are useful fortreatment of inflammation, which can result from a variety of ailmentssuch as, for example, asthma, allergies such as allergic rhinitis,uticaria, anaphylaxis, drug sensitivity, food sensitivity, etc. and thelike, cutaneous inflammation such as dermatitis, eczema, psoraisis,contact dermatitis, sunburn, aging, etc. and the like, and arthritissuch as osteoarthritis, psoriatic arthritis, lupus, spondylarthritis,etc. and the like. These peptides also are useful for treating chronicobstruction pulmonary disease and chronic inflammatory bowel disease.The peptides of the present invention can be used to replacecorticosteroids in any application in which corticosteroids are usedincluding immunosuppression in transplants and cancer therapy.

Continued mast cell degranulation and its release of leukotrienes,histamines, and other cytokines decreases, or ceases entirely inpreferred embodiments, following treatment with peptides of the presentinvention. In accord with preferred embodiments of the presentinvention, the peptides also can reduce the infiltration of eosinophils,basophils and neutrophils into inflammatory tissues. Lymphocytes,eosinophils, and neutrophils do not exhibit chemotaxis in response topreferred peptides of the present invention. As a consequence, thechemotactic adhesion, migration and aggregation of lymphocytes,eosinophils and neutrophils to the site of inflammation is significantlyreduced, as is vascular permeability at the inflammation site. Further,preferred compounds of the present invention exhibit no toxicity tovital organs such as heart, liver and lungs.

Preferred peptides, in accord with the present invention, provide areceptor link that blocks IgE activation of lymphocytes such as, forexample, macrophages, monocytes, eosinophils, neutrophils, TNF and thelike, in vitro and in vivo. The peptides stabilize the cell membrane ofsuch lymphocytes, preventing their further involvement in the increasedinflammatory response to an IgE antigen challenge. The peptides alsoblock cross-cell IgE linking in chronic inflammation, for example,between mast cells and eosinophils.

The peptides of this invention can be prepared by conventional smallpeptide chemistry techniques. The peptides when used for administrationare prepared under aseptic conditions with a pharmaceutically acceptablecarrier or diluent.

Doses of the pharmaceutical compositions will vary depending upon thesubject and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg a day, more preferably 1 to 10,000μg/kg. Most preferred dosages range from about 1 to 100 .μg/kg of bodyweight, more preferably from about 1 to 10 μg/kg and most preferably 1.0to 2.0 μg/kg. Doses are typically administered from once a day to every4-6 hours depending on the severity of the condition. For acuteconditions, it is preferred to administer the peptide every 4-6 hours.For maintenance or therapeutic use, it may be preferred to administeronly once or twice a day. Preferably, from about 0.18 to about 16 mg ofpeptide are administered per day, depending upon the route ofadministration and the severity of the condition. Desired time intervalsfor delivery of multiple doses of a particular composition can bedetermined by one of ordinary skill in the art employing no more thanroutine experimentation.

Routes of administration include oral, parenteral, rectal, intravaginal,topical, nasal, ophthalmic, direct injection, etc. In a preferredembodiment, the peptides of this invention are administered to thepatient in an anti-inflammatory effective amount or in a dosage thatinhibits degranulation of mast cells. An exemplary pharmaceuticalcomposition is a therapeutically effective amount of a peptide in accordwith the present invention that provides anti-inflammatory effect orthat inhibits degranulation of mast cells, typically included in apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” as used herein, anddescribed more fully below, includes one or more compatible solid orliquid filler diluents or encapsulating substances that are suitable foradministration to a human or other animal. In the present invention, theterm “carrier” thus denotes an organic or inorganic ingredient, naturalor synthetic, with which the molecules of the invention are combined tofacilitate application. The term “therapeutically-effective amount” isthat amount of the present pharmaceutical compositions, which produces adesired result or exerts a desired influence on the particular conditionbeing treated. Various concentrations may be used in preparingcompositions incorporating the same ingredient to provide for variationsin the age of the patient to be treated, the severity of the condition,the duration of the treatment and the mode of administration.

The carrier must also be compatible. The term “compatible”, as usedherein, means that the components of the pharmaceutical compositions arecapable of being comingled with a small peptides of the presentinvention, and with each other, in a manner such that does notsubstantially impair the desired pharmaceutical efficacy.

The small peptides of the invention are typically administered per se(neat). However, they may be administered in the form of apharmaceutically acceptable salt. Such pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene-sulfonic, tartaric, citric,methanesulphonic, formic, malonic, succinic, naphthalene-2-sulfonic, andbenzenesulphonic. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group. Thus, thepresent invention provides pharmaceutical compositions, for medical use,which comprise peptides of the invention together with one or morepharmaceutically acceptable carriers thereof and optionally any othertherapeutic ingredients.

The compositions include those suitable for oral, rectal, intravaginal,topical, nasal, ophthalmic or parenteral administration, all of whichmay be used as routes of administration using the materials of thepresent invention. Pharmaceutical compositions containing peptides ofthe present invention may also contain one or more pharmaceuticallyacceptable carriers, which may include excipients such as stabilizers(to promote long term storage), emulsifiers, binding agents, thickeningagents, salts, preservatives, solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with thepeptide of this invention, its use in pharmaceutical preparations iscontemplated herein. Supplementary active ingredients can also beincorporated into the compositions of the present invention.

Compositions suitable for oral administration are preferred fortreatment of asthma. Typically, such compositions are prepared as aninhalation aerosol, nebule, syrup or tablet. Compositions suitable fortopical administration are preferred for treatment of arthritis,although oral compositions also can be convenient. Typically, suchtopical compositions are prepared as a cream, an ointment, or asolution. The concentrations of the peptide active ingredient in suchcompositions is typically less than 50.μg/ml, more preferable less than30 μg/ml, and most preferably from about 5 to 10 μg/ml.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Methods typically include the step of bringing the active ingredients ofthe invention into association with a carrier that constitutes one ormore accessory ingredients.

Compositions of the present invention suitable for inhalationadministration may be presented, for example, as aerosols or inhalationsolutions. An example of a typical aerosol composition consists of thedesired quantity of microcrystalline peptide suspended in a mixture oftrichloromonofluoromethane and dichlorodifluoromethane plus oleic acid.An example of a typical solution consists of the desired quantity ofpeptide dissolved or suspended in sterile saline (optionally about 5%v/v dimethylsulfoxide (“DMSO”) for solubility), benzalkonium chloride,and sulfuric acid (to adjust pH).

Compositions of the present invention suitable for oral administrationalso may be presented as discrete units such as capsules, cachets,tablets or lozenges, each containing a predetermined amount of thepeptide of the invention, or which may be contained in liposomes or as asuspension in an aqueous liquor or non-aqueous liquid such as a syrup,an elixir, or an emulsion. An example of a tablet formulation baseincludes corn starch, lactose and magnesium stearate as inactiveingredients. An example of a syrup formulation base includes citricacid, coloring dye, flavoring agent, hydroxypropylmethylcellulose,saccharin, sodium benzoate, sodium citrate and purified water.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the molecule of the invention,which is preferably isotonic with the blood of the recipient. Thisaqueous preparation may be formulated according to known methods usingthose suitable dispersing or vetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In aqueous solutions, up to about10% v/v DMSO or Trappsol can be used to maintain solubility of somepeptides. Also, sterile, fixed oils may be conventionally employed as asolvent or suspending medium. For this purpose, a number of fixed oilscan be employed including synthetic mono- or diglycerides. In addition,fatty acids (such as oleic acid or neutral fatty acids) can be used inthe preparation of injectibles. Further, Pluronic block copolymers canbe formulated with lipids at 4° C. for compound injection on a timerelease basis from solid form at 37° C. over a period of weeks ormonths.

Compositions suitable for topical administration may be presented as asolution of the peptide in Trappsol or DMSO, or in a cream, ointment, orlotion. Typically, about 0.1 to about 2.5% active ingredient isincorporated into the base or carrier. An example of a cream formulationbase includes purified water, petrolatum, benzyl alcohol, stearylalcohol, propylene glycol, isopropyl myristate, polyoxyl 40 stearate,carbomer 934, sodium lauryl sulfate, acetate disodium, sodium hydroxide,and optionally DMSO. An example of an ointment formulation base includeswhite petrolatum and optionally mineral oil, sorbitan sesquioleate, andDMSO. An example of a lotion formulation base includes carbomer 940,propylene glycol, polysorbate 40, propylene glycol stearate, cholesteroland related sterols, isopropyl myristate, sorbitan palmitate, acetylalcohol, triethanolamine, ascorbic acid, simethicone, and purifiedwater.

The Rat Skin Model for Determination of Inhibition of Mast CellDegranulation

Allergy induced asthma results from exposure of substances (allergens)to which an organism has become hypersensitized. Exposure to allergenresults in degranulation of mast cells in the lung, releasingleukotrienes and histamines. In response to the release of leukotrienesand histamines, capillary permeability is dramatically increased andblood plasma leaks from the capillaries into the surrounding tissues.Respiratory symptoms resulting from such an exposure range from mild(itching and sneezing) to potentially fatal (asthma), including inextreme chronic cases death by anaphylaxis.

To demonstrate this phenomenon experimentally, rat skin is substitutedfor lung. In this model, the blood plasma of the experimental rat islabeled with the dye trypan blue. This soluble dye is carried in thebloodstream as a passive marker of plasma itself, and is excluded fromlive cells. Intact blood vessels, including the capillary system, retainthis dye under normal circumstances. A compound, which inducesdegranulation of mast cells (resulting in leukotriene and histaminerelease), is injected into the skin to simulate allergen-induceddegranulation. In these experiments, Compound 48/80 was used for thispurpose. In the events following leukotriene and histamine release,capillary permeability is increased, and plasma, dyed blue, leaks fromcapillaries and dyes the skin surrounding the injection site blue. Thearea of bluing is a measure of the amount of Compound 48/80 injected.

A compound can be tested for “anti-leukotriene” and/or “anti-histamine”activity by mixing it with Compound 48/80 prior to injection. If thetest compound inhibits leukotriene or histamine release, an area ofbluing of smaller diameter is observed when compared to an injectionsite on the same rat into which Compound 48/80 has been injected withoutany of the test compound. In the case of high anti-leukotriene andanti-histamine activity, the bluing may actually be totally inhibited.

EXPERIMENTAL

The rat skin model was undertaken and validated. Various peptides weretested at a predetermined dose for anti-leukotriene and/oranti-histamine activity. The dose selected allowed a general comparisonto f-Met-Leu-Phe, which was standard compound for comparison.

A “dose response” titration was performed for some compounds andcompared with the standard compound. Observing serial decreases in thesize areas of capillary permeability using serially smaller doses of theputative inhibitory compound validates the inhibition of leukotrieneand/or histamine release observed in the initial predetermined dosetest.

Materials and Methods

Reagents were obtained from Sigma or Aldrich, with the exception ofketamine, a veterinary anaesthetic that was obtained from variousveterinary suppliers. The rats used were male Sprague-Dawley breed,220-240 g at time of purchase from B&K International.

For the rat skin reaction, rats were anaesthetized with 0.25 ml 10 mg/mlketamine. 1.0 ml trypan blue in saline (sterile filtered) wasadministered in a tail vein, and the back of the rat was shaved. Fourintradermal injection sites per rat were used for test and controlinjections.

Compound 48/80 was prepared as a 1.5 mg/ml stock solution in saline.This material was found to be potentially unstable in aqueous solutionand was prepared freshly each day. Serial dilutions in saline to workinglevels were prepared just prior to injection of each rat.

Peptides were prepared as a 23 mM stock solution in DMSO, and stored at−20° C. between experiments. At the time of use, the frozen stocksolutions were thawed, and appropriate aliquots added to dilutions ofCompound 48/80, along with appropriate amounts of DMSO, to result in theratio of 5 μl DMSO to 0.1 ml aqueous Compound 48/80. This resulted in a5% solution of DMSO, necessary to maintain solubility of certainpeptides. The effect of 5% DMSO was demonstrated by control experimentsto be nil.

For injections, 0.1 ml Compound 40/80, +1/− test compounds were injectedintradermally into anaesthetized, dyed, and shaved rats. Following a 15minute incubation, the rats were sacrificed by cervical dislocation andthe back skin was evulsed and placed on a light box. An image of thebacklit skin was digitized using a CCD video capture camera andcompatible hardware/software. The digitized image was analyzed using ascientific graphics analysis software package, and the areas ofcapillary permeability (bluing) were integrated and digital values wereobtained for further analysis.

A dose response curve was generated using Compound 48/80 at variousdoses from ca. 0.01.1 g through ca. 15 μg. The results are shown inFIG. 1. Wide variability was noted in the diameter of areas of capillarypermeability for a given dose of Compound 48/80 based upon rat-to-ratvariations (e.g., thickness of skin). A dose of 0.15 μg of Compound48/80 was selected for conducting further tests.

Example 1

A dose response curve was prepared for the standard compound,f-Met-Leu-Phe, using the selected dose of 0.15 μg Compound 48/80. Dosesof 0 to about 230 nM of f-Met-Leu-Phe were tested and the results areshown in FIG. 2. Inhibition of degranulation induced by Compound 48/80was clearly shown.

Examples 2-11

Several f-Met-Leu peptides were tested for inhibition of induceddegranulation in the rat skin model using 100 nanomoles of the testpeptide and a dose of 0.15 μg Compound 48/80. An intrinsiczero-peptide-dose 48/80 control was included in each rat for eachexperiment, and the % of inhibition was expressed in relative terms tothis control (0% inhibition). The percent mast cell degranulationproduced by 48/80 was also determined. The results are tabulated below.

TABLE 1 % % SEQ Inhibi- Degranu- Example Peptide ID tion lation* 2f-Met-Leu-Phe (prior 30 60 art) 3 N-acetyl-Met-Leu-Phe 0 98 4N-t-BOC-Met-Leu-Phe 0 — 5 f-Met-Leu-(iodo)Phe 0 — 6 f-Met-Leu-Phe 0 —(benzylamide) 7 f-Met-Leu-Phe-Lys 4 0 — 8 f-Met-Leu-Phe(methyl 0 —ester) 9 f-Met-Leu-Phe-Phe 2 100 1-3 10 f-Met-Leu-Tyr 55 30 11f-Met-Leu-Tyr-Tyr 5 0 —

Example 12

A dose response curve was prepared for f-Met-Leu-Phe-Phe using theselected dose of 0.15 μg Compound 48/80. Doses of 0 to about 230 nM off-Met-Leu-Phe-Phe were tested and the results are shown in FIG. 3.Surprisingly remarkable inhibition of degranulation induced by Compound48/80 was clearly shown. The inhibition of induced degranulation forf-Met-Leu-Phe-Phe was unexpectedly substantially better than that of thestandard compound f-Met-Leu-Phe.

The OVA-induced Bronchial Asthma Mouse Model for Inhibition of Mast CellDegranulation

Asthma is a complex disease, which is characterized by spontaneousexacerbation of airways obstruction and persistent bronchialhyperresponsiveness. Chronic infiltration with activated T-lymphocytes,eosinophils and macrophages/monocytes of the airway submucosa is anotherestablished feature. Inflammatory mechanisms, with expression ofcytokines, and the release of inflammatory mediators, underlie thepathogenesis of bronchoconstriction and bronchial hyperresponsiveness.However, much of the pathogenic mechanism remains unclear, e.g., themechanisms that induce persistence of symptoms and chronic inflammationand the interventions necessary to control and prevent the disease.

It has long been recognized that a single inhaled allergen challenge caninduce an acute increase in airway responsiveness in some individualsand animal models. However, repeated allergen inhalations havedemonstrated more pronounced, consistent, and prolonged increases inairway responsiveness. This mouse model of long-term repeatedinhalations of allergen has been used to study the long term effect ofallergic diseases in the lung, and to delineate the cells, mechanisms,molecules, and mediators involved in the induction of airwayhyperresponsiveness of lung in humans.

Materials and Methods

Reagents: Crystalline OVA was obtained from Pierce Chem. Co. (Rockford,Ill.) aluminum potassium sulfate (alum) from Sigma Chem. Co. (St. Louis,Mo.), pyrogen-free distilled water from Baxter, Healthcare Corporation(Deerfield, Ill.), 0.9% sodium chloride (normal saline) from Lymphomed(Deerfield, Ill.) and Trappsol.™. HPB-L100 (aqueous hydroxypropyl βcyclodextrin; 45 wt/vol % aqueous solution) from CyclodextrinTechnologies Development, Inc. (Gainesville, Fla.). The OVA (500 .μg/mlin normal saline) was mixed with equal volumes of 10% (wt/vol) alum indistilled water. The mixture (pH 6.5 using 10 N NaOH) after incubationfor 60 minutes at room temperature underwent centrifugation at 750 g for5 minutes; the pellet was resuspended to the original volume indistilled water and used within one hour.

The selective 5-lipoxtgenase inhibitor, Zileuton(N-[1-benzo[b]thien-2-ylethyl]-N-hydroxyurea; J. Pharmacol Exp Ther.1991; 256: 929-937), was kindly provided by Drs. Bell and George W.Carter (Abbott Laboratories, Abbott Part, Ill.). Zileuton was dissolvedin Trappsol.™. Histatek, Inc. (Seattle, Wash.) provided the mast celldegranulation inhibitor, f-Met-Leu-Phe-Phe (“HK-X”).

Female BALB/c Once (6-8 wk of age at purchase; D and K, Seattle Wash.)were housed under conventional conditions for the studies.

Allergen Immunization/Challenge Protocols: Mice received an i.p.injection of 0.2 ml (100 μg) of OVA with alum on the different protocolsof Standard (FIG. 5A) and Resolution (FIG. 5B) (J. Exp Med. 1996; 184:1483-1494). According to the different protocols, mice were anesthetizedwith 0.2 ml i.p. of ketamine (0.44 mg/ml)/xylazine (6.3 mg/ml) in normalsaline before receiving an intranasal (i.n.) dose of 100 μg OVA in 0.05ml normal saline and an i.n. dose of 50.μg OVA in 0.05 ml normal salineseparately on different days. Two control groups were used. Accordingly,the first group received normal saline with alum i.p. and normal salinewithout alum i.n.; the second group received OVA with alum i.p., OVAwithout alum i.n., and normal saline, alone.

Histology

The trachea and left lung (the right lung is used for bronchoalveolarlavage (“BAL”)) were obtained and fixed in 10% neutral formaldehydesolution at room temperature for 6 about 15 h. After being embedded inparaffin, the tissues were cut into 5-um sections and processed with thedifferent staining or immunolabling further. Discombe's eosinophilstaining was used for counting the cell numbers with the counterstain ofmethylene blue. The eosinophil number per unit airway area (2,200 μm.²)was determined by morphometry (J. Pathol. 1992; 166: 395-404; Am RevRespir Dis. 1993; 147:448-456). Fibrosis was identified with theMasson's trichrome staining. Airway mucus was identified by thefollowing staining method: methylene blue, hematoxylin and eosin,mucicarmine, alcian blue, and alcian blue/periodic acid-Schiff (PAS)reaction (Troyer, H., “Carbohydrates” in Principles and Techniques ofHistochemistry, Little, Brown and Company, Boston, Mass., 1980: 89-121;Sheehan, D.C., et al., “Carbohydrates” in Theory and Practice ofHistotechnology, Battle Press, Columbus, Ohio, 1980: 159-179) Mucin wasstained with mucicarmine solution; metanil yellow counterstain wasemployed. Acidic mucin and sulfated mucosubstances were stained withalcian blue, pH 2.5; nuclear fast red counterstain was used. Neutral andacidic mucosubstances were identified by alcian blue, pH 2.5, and PASreaction. The degree of mucus plugging of the airways (0.5-0.8 mm indiameter) was also assessed by morphometry. The percent occlusion ofairway diameter by mucus was classified on a semiquantitative scale from0 to 4+ as described in Figure Legends. The histologic and morphometricanalyses were performed by individuals blinded to the protocol design.

Pulmonary Function Testing

On day 28, 24 hours after the last i.n. administration of either normalsaline or OVA, pulmonary mechanics to intravenous infusion ofmethacholine were determined in mice in vivo by a plethysmographicmethod, which was modified from that previously described (10, 1958;192: 364-368; J. Appl. Physiol. 1988; 64: 2318-2323; J. Exp. Med. 1996;184: 1483-1494). At the completion of pulmonary function testing, eachmouse was exsanguinated by cardiac puncture and the lung tissue withtrachea was obtained for the further analysis.

Bronchoalveolar Lavage

After tying off the left lung at the mainstem bronchus, the right lungwas lavaged three times with 0.4 ml of normal saline. Bronchoalveolarlavage (BAL) fluid cells from a 0.05-ml aliquot of the pooled samplewere counted sing a hemocytometer and the remaining fluid centrifuged at4° C. for 10 minutes at 200 g. The supernatant was stored at 70° C.until eicosanoid analysis was performed. After resuspension of the cellpellet in normal saline containing 10% bovine serum albumin (“BSA”), BALcell smears were made on glass slides. To stain eosinophils, driedslides were stained with Discombe's diluting fluid (0.05% aqueous eosinand 5% acetone (vol/vol) in distilled water; J. Exp. Med. 1970; 131:1271-1287) for 5-8 minutes, rinsed with water for 0.5 minutes, andcounterstained with 0.07% methylene blue for 2 minutes.

Assay of Airway Mucus Glycoproteins

Mucus glycoproteins in BAL fluid were assayed by slot blotting and PASstaining (Anal. Biochem. 1989; 182: 160-164; Am. J. Respir. Cell Mol.Biol. 1995; 12: 296-306). Nitrocellulose membranes (0.2-μ pore size;Schleicher & Schuell, Keene, N.H.) were wetted in distilled water andthen in normal saline before placement in a Minifold II 72-well slotblot apparatus (Schleicher & Schuell). The BAL fluid samples (0.05 ml)and aliquots (0.05-0.75 l) of a stock solution (2 μm/ml) of humanrespiratory mucin glycoprotein (Am. J. Respir. Cell Mol. Biol. 1991; 5:71-79) were blotted onto the nitro-cellulose membranes by water suctionvacuum, and mucus glycoproteins were visualized by PAS reaction.Reflectance densitometry was performed to quantitate the PAS staining.The images were than analyzed by an image processing system describedbelow. The integrated intensity of the PAS reactivity of the BAL sampleswas quantitated by comparison to the standard curve for humanrespiratory mucin.

Immunocytochemistry

Monoclonal antibody: CD 11c (DAB method) and Mac1 (Beringer Mannheim,ABC method with Hitomouse Kit, Zymed) were used to identified theinflammatory cell types, e.g., dendric cells, macrophages andlymphocytes, in/around the areas of vasculatures, airways and fibrosis.

Morphometry and Image Analysis

All the images were captured and digitized by a ScanJet IICX Scannerwith HP DeskScan II software (Microsoft.®. Windows.™. Version) (HewlettPackard, Palo Alto, Calif.). This system was linked to Dell DimensionXPS P90 computer (Dell Corporation, Austin, Tex.) employing Image-Pro.®.Plus, version 1.1 for Windows.™. software (Media Cybernetics, SilverSpring, Md.). The images were assessed on a 256 gray level scale using aDell Ultrascan 17ES monitor with extra high-resolution graphics mode(1.280.times.1,024 pixels, 78.9-kHz horizontal scanning frequency, 74-Hzvertical scanning frequency).

Leukotriene Inhibitor Studies

To assess the role of 5-lipoxygenase products in airway inflammation,the 5-lipoxygenase inhibitor, Zileuton, (35 mg/kg) was given i.p. 30minutes before each i.n. challenge on the days according to FIG. 5. Inone set of animals, Zileuton was also given before i.p. OVA. Zileuton at35 mg/kg inhibits cysteinyl leukotriene release by about 95% inpassively sensitized rats given BSA antigen i.p. (J. Pharmacol. Exp.Ther. 1991; 256: 929-937).

Compound HK-X of the Invention

Compound HK-X was administered at 5 mg/kg and 10 mg/kg using the sameprocedure as described above.

Statistical Analyses

The pulmonary function data were evaluated by analysis of variance(ANOVA) using the protected least significant difference method(Statview II, Abacus Concepts, Berkeley, Calif.). This method uses amultiple t statistic to evaluate all possible pairwise comparisons andis applicable for both equal and unequal pair sizes. The other data arereported as the mean.+−.SE of the combined experiments. Differences wereanalyzed for significance (P<0.05) by Student's two-tailed t test forindependent means.

1. Eosinophils (Tables 2A-2B)

The eosinophil numbers of the airway in OVA-treated mouse of 1-, 2- and3-month group were significantly reduced from 44-83% to 37.40% and19.15%, respectively (P<0.025). Even though the eosinophil count is muchhigher in the OVA treated group than the other two groups at the sametime course (P<0.025), Zileuton could reduced eosinophils generallythrough 1-3 month. However, the HK-X compound of the present inventionreduced eosinophils comparably at one month, but much more beneficiallyat two and three months.

TABLE 2A AIRWAY INFLUX OF EOSINOPHILS (%) Saline OVA Zileuton HK P value3 month 1.00 19.15 10.73 — <0.025 2 month 1.00 37.40 11.66 — <0.01 1month 1.00 44.83 15.50 14.20 <0.001 P value >0.05 <0.025 <.025 <0.025

TABLE 2B PERCENTAGE OF EOSINOPHILS IN AIRWAY TISSUE Time of TreatmentSaline OVA Zileuton HK-X 28 days 1.0 44.8 15.8 14.2

2. Other Inflammation Cells

Other inflammation cells indicate a non-specific inflammatory responsefollowing the introduction into the airway of a foreign protein.Lymphocytes were recruited into the airways, but were virtually absentin control groups. Neutrophils were recruited following OVA challenge inboot sham-sensitized and OVA-sensitized mice, although greater numberswere presented in the airways of the OVA sensitized group. Peculiarmultinucleate giant cells (fused macrophages) having crescents of nucleiaround the periphery of their extensive cytoplasm, were occasionallyseen. Both Langhans giant cells and globule leukocytes were observedonly in animals sensitized and challenged with OVA. They were usuallypresent in the connective tissue associated with larger airways. Plasmacells were occasionally seen in the proximity of the airways and inlocal lymphoid tissue.

3. Airway Plug (Table 3)

Mucin: There was no difference among the three groups with the sametreatment but difference time course (P>0.05). The OVA-treated group hada higher score than that of the groups treated with saline, Zileuton(P<0.05) and HK-X compound.

TABLE 3 MUCUS PLUG SCORE IN AIRWAYS Time of treatment Saline OVAZileuton HK-X 28 days 0.7 2.8 1.3 1.4 % of plug of airway >5% 55% 16%19%Asthma is a chronic inflammatory condition of the airways. In humans,once it is established, the airway hyperresponsiveness can remain stablefor years. It persists apparently in the absence of allergen inhalation,detectable airway inflammation or epithelian desquamation. Thus, it maybecome permanent due to irreversible (or at least slowly reversible)alterations in airway ultrastructure.

In mild asthmatics, these episodes or “attacks” are relativelyinfrequent and well-treated (reversed) with inhaled bronchodilators. Itsintensity of an underlying, distinctive and chronic airway inflammationis associated, and seemingly linked, to more frequent, intense andprolonged attacks that are less reversible by bronchodilators. Thereasons for this have become increasingly clear in recent years. Theinflammation, which consists principally of an activated or primedinfiltrate of Th₂-lymphocytes, eosinophils, mast cells, and possiblyplatelets, causes an expansion of the perivascular ((interstitial)spaces and release of mediators/growth factors, which cause thickeningof the basement membrane, epithelial damage and shedding, production ofviscous mucus, and hyperplasia, priming as well as partial constrictionof airway smooth muscle. All of these outcomes support an increase inairway responsiveness, which lowers the threshold for response toenvironmental stimuli, thus making attacks more frequent and robust.

All the above morphological changes will directly and strongly affectthe pulmonary functions. In experiments on acute asthmatic mouse modeland on long-term asthmatic mouse model, the significantpathophysiological changes of pulmonary functions have been observed tosupport the above morphological changes. Allergen inhalation was foundto increase eosinophils and mast cells expression on airway and alveolarendothelium and epithelium, as well as inducing E-selection expressiononly on airway endothelium, and both the eosinophil infiltration andincrease in airway responsiveness, and the other types of inflammatorycells (globule leukocytes and multinucleate giant cells (fusedmacrophages) of the Langhans type), which indicated non-specificinflammatory reaction within the asthmatic lungs.

Compound HK-X inhibits mucus accumulation in the airway of OVA-treated(OVA) and control mice. The distribution of mucus occlusion of airwayswas determined from sham-sensitized and saline-challenged mice (saline,n=4), and OVA-sensitized/challenged mice in the absence (OVA, n=4) orpresence (HK-X/OVA, n=8) of HK-X treatment. Mucus occlusion of airwaydiameter was assayed morphometrically as following: 0, no mucus; +,about 10% occlusion; ++, 30% occlusion; +++, about 60% occlusion; ++++,about 80% occlusion. 10 airways randomly distributed throughout thelungs of each mouse were assessed for mucus occlusion morphometrically.

FIGS. 6A-6D provide visual histologic evidence of the ability to ofCompound HK-X to inhibit degranulation of mast cells in asthma inducedrats using OVA and thereby the effect of treating asthma with CompoundHK-X. FIG. 6A shows an abundance of secreted mucus in the lumen of theairway (AW) of OVA sensitized/challenged mice. FIG. 6B shows massiveinfiltration of the interstitial tissue by eosinophils and otherinflammatory cells (noted by arrows). FIG. 6C shows that airway mucusrelease in the airway (AW) lumen is markedly reduced when Compound HK-Xinhibitor is given before i.n. OVA. The infiltration of the interstitialtissue by eosinophils is also reduced after Compound HK-X treatmentcompared to OVA-challenge alone (compare FIG. 6C with FIGS. 6A and 6B).FIG. 6D shows that the airway (AW) is clear of mucus and cells inSaline-treated control mice. The bronchial epithelium is infiltratedwith connective tissue cells but no leukocytes are present in theperibronchial interstitial space.

Airway macrophages showed signs of gross activation that resembled thosereported in macrophages recovered in BAL fluid from allergen-challengedlungs of asthmatics (Am. Rev. Respir. Dis. 1987; 135: 433-440).Macrophages and dendritic cells function as antigen-presenting cells inlung and may lead, directly or indirectly, to the secretion of cytokinesable to initiate phenotypic changes in airway epithelium and itsperipheral sites. The stimulation of the chronic inflammation of theairway may directly induce the proliferation of airway epithelium andfibroblasts, and the consequent collagen deposit around these areas.Activated macrophages and dendric cells remained high in the area incomparison with the other inflammatory cells during the late-stagechallenges.

The airway epithelium was thickened, due largely to a marked goblet cellhyperplasia, particularly in the larger airways, but also in small andeven terminal bronchioles. The ratio of goblet cells to normal,columnar, ciliated cells was greatly increased compared with controlgroups. Whereas control airways (both small and large) had only theoccasional goblet cells, section from OVA-challenged lungs showed that100% of large airways and part of small airways contained goblet cellsas up to 88% of the total airway epithelial cells. In lungs that had notbeen lavaged, mucus could be seen within the goblet cell and in someairways, occasionally completely occluding the lumen. Cellular debriswas enmeshed in these mucus plugs. Goblet cell hyperplasia was not seenin control groups and, therefore, could not have been due to a“non-allergic” effect of OVA, or to the intratracheal dosing technique.Some of the goblet cells in the small airways are free of this feature,indicating perhaps that the distribution of OVA within the respiratorytree had not been uniform.

FIG. 7 is a histogram of the results for treatment with Compound HK-X atdoses of 5 μg/kg and 10 μg/kg on formation of mucus plugs in this murineasthma model. Both doses significantly reduce the mucus production insmall airways.

Asthma is characterized by a complex inflammatory response of airwayeosinophilia, edema, mucus hypersecretion, bronchial epithelial injuryand hyperreactivity. Inhaled allergen challenge in allergic asthmaticsprovokes an immediate airway hypersensitivity reaction, an early airwayresponse (EAR), that is frequently followed several hours later by adelayed airway reaction, a late phase airway response (LAR). Afterrecovery from LAR, there is an increase in acquired airwayhyperreactivity (AHR) to agents such as methacholine that may persistfor several days. The EAR occurring shortly after allergen challenge islikely secondary to the action of bronchoconstrictor molecules releasedby human lung mast cells as a consequence of IgE-mediated degranulation.

IgE-mediated mast cell degranulation is the primary event in thepathogenesis of such allergic disorders as allergic rhinitis, asthma,and anaphylaxis. In atopic individuals, the intracutaneous injection ofspecific allergen results in an immediate wheel and flare response thatis characterized by the release of histamine and formation of lipidmediators at the skin test site. The early skin response is followed bya late skin reaction occurring 6 to 12 hours later. This late allergicskin reaction is characterized by an inflammatory response consisting ofperivascular edema and cellular infiltration by eosinophils and otherinflammatory cells (e.g., neutrophils, monocytes, and basophils).Similar dual phase IgE dependent reaction such as, early and laterhinitis or asthmatic responses occurs in the upper and lower airways ofatopic individuals after local challenge with specific allergen. Mastcells are located in close proximity to the alveolar surface to bloodvessels. Mast cells may influence the pulmonary vasculature by affectingtone or by promoting an inflammatory response. Mast cells activated byimmunologic or non-immunologic stimuli degranulate and release amultitude of preformed and newly generated mediators such as histamine,neutral proteases, peroxidase, O₂, PAF and eicosanoids (e.g., LTB4,LTC4, PGD2, TXA2) and cytokines (e.g., IL-4, IL-5, TNFα), which maymediate lung inflammation.

The above-described murine model reproduces key features of humanasthma. Late-phase allergen-specific pulmonary disease was induced innormal BALB/c mice using ovalbumin (OVA) as allergen. One protocolincludes immunization of mice with i.p. OVA on days 1 and 14, andintranasal (i.n.) administration of OVA on days 14, 25, 26, and 27. Onday 28, OVA-treated mice display a disease strikingly similar toallergen induced asthma including: (1) increased circulating levels oftotal and OVA-specific IgE, (2) increased release of LTB4 and LTC4 inBAL fluid, (3) a marked eosinophil influx into BAL fluid and thepulmonary parenchyma, (4) mucus occlusion of small airways, (5)increased expression of T-helper cell type 2 (Th₂) cytokines (IL-4,IL-5, and IL-13) and decreased expression of Th₁ cytokines (IL-2 andIFN-Y) in bronchial lymph node tissue, (6) pulmonary hyperreactivity, asassessed by a significantly more rapid decline in airway conductance anddynamic compliance with increasing doses of methacholine compared tocontrol mice.

The mouse is also susceptible to development of IgE-mediated allergicairway responses. The late-phase influx of eosinophils is reproduced inthis murine model in which allergic airway disease develops afterovalbumin inhalation in mice previously sensitized to ovalbuminintraperitoneally. Increased airway responsiveness to methacholine oracetylcholine challenge also occurs in immunized mice following airwayexposure to antigen.

In a mouse model of allergen-induced airway inflammation, the role ofmast cell in airway eosinophil infiltration, mucus release, andhyper-responsiveness to methacholine was examined. The small peptideHK-X was found to be a key to the prevention of the mucus release by themechanism of prevention of mast cell degranulation and eosinophilinfiltration of the airways.

Materials were used as described above. Mice received an i.p. injectionof 0.2 ml (100 ug) of OVA complexed with aluminum potassium sulfate(alum) on day 0 and 14. On days 14, 25, 26, and 27, mice wereanesthetized with 0.2 ml i.p. of ketamine (0.44 mg/ml)/xylazine (6.3mg/ml) in normal saline before receiving an intranasal (i.n.) dose of100 ug OVA in 0.05 ml normal saline on days 25, 26, and 27. Lung tissuewas obtained 24 hours after the last i.n. challenge on day 28. Thecontrol group received normal saline with alum i.p. on days 0 and 14 andnormal saline without alum i.n. on days 14, 25, 26, and 27. To assessthe effect of HK-X inhibition on OVA induced asthma, HK-X (10 μg/ml) wasgiven i.n. 30 minutes before each i.n challenge on days 25, 26, and 27.

After tying off the left lung at the mainstem bronchus, the right lungwas ravaged three times with 0.4 ml of normal saline, Bronchoalveolarlavage (BAL) fluid cells from a 0.05 ml aliquot of the pooled samplewere counted using a hemocytometer and the remaining fluid centrifugedat 4° C. for 10 minutes at 200 g. After resuspension of the cell pelletin normal saline containing 10% BSA, BAL cell smears were made on glassslides. To stain eosinophils, dried slides were stained with Discombe'sdiluting fluid (0.05% aqueous eosin and 5% (v/v) acetone in distilledwater) for 5-8 minutes, rinsed with water for 0.5 minutes, andcounterstained with 0.07 methylene blue for 2 minutes.

Following BAL, the trachea and left lung (upper and lower lobes) wereobtained and fixed in 10% buffered formalin solution at 20° C. for IShours. After the tissues were processed and embedded in paraffin, thetissues were cut into 5 μm sections and stained with Discombe's solutionand counterstained with methylene blue as described above or stainedwith Hematoxylin and eosin. The eosinophil number per unit airway area(2200 μm²) was determined by morphometry as previously described. Airwaymucus was identified by a variety of staining methods, i.e., methyleneblue, Hematoxylin and eosin, mucicarmine, toluidine blue, alcian blue,and alcian blue/periodic acid Schiff (PAS) reaction. Mucin was stainedwith mucicarmine solution; metanil yellow counterstain was employed.Mucin and sialic acid-rich non-sulfated mucosubstances were stainedmetachromatically with toluidine blue, pH 4.5. Acidic mucin and sulfatedmucosubstances were stained with alcian blue, pH 2.5; nuclear fast redcounterstain was used. Neutral and acidic mucosubstances were identifiedby alcian blue, pH 2.5 and PAS reaction. The degree of mucus plugging ofthe airways (0.5.about .0.8 mm in diameter) also was assessed bymorphometry. The percent occlusion of airway diameter by mucus wasclassified on a semiquantitative scale from 0 to +++++. The histologicand morphometric analyses protocol design were performed by individualsblinded to the protocol design.

Mucus glycoproteins in BAL fluid were assayed by slot blotting and PASstaining as described. Nitrocellulose membranes (0.2 um pore size;Schleicher & Schuell, Keene, N.H.) were wetted in distilled water andthen in normal saline before placement in a Minifold II 72-well slotblot apparatus (Schleicher & Schuell). The BAL fluid samples (0.05 ml)and aliquots (0.05-0.75 ml) of a stock solution (2 ug/ml) of humanrespiratory mucin glycoprotein were blotted onto the nitrocellulosemembranes by water suction vacuum, and mucus glycoproteins werevisualized by PAS reaction. Reflectance densitometry was performed toquantitate the PAS staining. The images were captured and digitized by aScanJet IIcx Scanner with HP DeskScan II software (Microsoft Windows™Version) (Hewlett Packard, Palo Alto, Calif.). This system was linked toa Dell Dimension XPS P90 computer (Dell Corporation, Austin, Tex.)employing Image-Pro Plus, Version 1.1 for Windows™ software (MediaCybernetics, Silver Spring, Md.). The images were assessed on a 256 graylevel scale using a Dell UltraScan 1 7ES monitor with extrahigh-resolution graphics mode (1280×1024 pixels, 78.9-kHz horizontalscanning frequency, 74-Hz vertical scanning frequency). The integratedintensity of the PAS reactivity of the BAL samples was quantitated bycomparison to the standard curve for human respiratory mucin.

I.p. immunization with OVA results in detectable levels of OVA-specificIgE in the blood of BALB/C mice. Indirect ELISA was employed todetermine OVA-specific IgE serum antibody titers. ELISA plates (ICN,Costa Mesa, Calif.) were coated with OVA (20 mg/ml) diluted in 0.1 MNAHCO, buffer pH 8.3 and incubated at 4 degrees for 18 hours. Afterwashing three times, the plates were incubated with 1% BSA in PBS, pH7.4, at 37° C. for 2 hours. Serial dilution of the serum samples in 1%BSA/PBS buffer were added to the plates and incubated at 4° C. for 18hours before washing again. The wells were incubated with HRP (horseradish peroxidase) conjugated rat anti-mouse IgE monoclonal antibody(Pharmingen, San Diego, Calif.) diluted in 50% goat serum (Gibco-BRL,Gaithersburg, Md.)/PBS buffer for 2 hours at room temperature.3,3′5,5′-tetramethylbenzidine substrate was used to develop the wellswith absorbance determined at 610 nm. The internal standard in eachassay consisted of pooled serum from OVA-immunized BALB/c mice.

The pulmonary function data were evaluated by analysis of variance(ANOVA) using the protected least significant difference method(Statview II, Abacus Concepts, Berkeley, Calif.). This method uses amultiple t-statistic to evaluate all possible pairwise comparisons andis applicable for both equal and unequal pair sizes. The other data arereported as the mean +SE of the combined experiments. Differences wereanalyzed for significance (p<0.05) by Student's two-tailed t-test forindependent means.

Results

Allergen-Specific IgE Production. OVA-specific IgE (12.9+0.3 U/ml, n=5)was detected on day 28 in the blood of mice given i.p. OVA and alum onday 0 and 14 and i.n. OVA on day 25, 26, and 27. In contrast, controlmice treated with i.p. saline and alum and i.n. saline (n=6) had nodetectable anti-OVA IgE.

Allergen-induced Airway Inflammation. To assess allergen-induced airwayinflammation histologically, lung tissue and BAL fluid were obtained onday 28, 24 hours after the last of 3 sequential i.n. OVA challenges ondays 25, 26, and 27. By light microscopy, prominent infiltration of thebronchial interstitium by eosinophils was observed (FIG. 8C). Eosinophilinflux into the bronchial epithelial mucus layer (FIG. 9D) and the BALfluid was also noted.

61.0+5.0% (n=10) of the BAL fluid cells were eosinophils inOVA-sensitized/challenged mice compared to 0.8+0.3% (n=10) insaline-challenged control animals (p=0.0001).

Mucus occlusion of the airways occurred in immunized mice afterbronchial challenge with OVA (FIG. 10). Airway mucus release in bothlower (FIG. 9B) and upper (FIGS. 9C, 9D) pulmonary airways wasidentified by separate histochemical staining procedures: mucin bymucicarmine stain, acidic non sulfated mucosubstances by toluidine blue,acidic sulfated mucosubstances by alcian blue (FIG. 10), and neutral andacidic mucosubstances by alcian blue/PAS reaction. Airway lumenocclusion by mucus was greater in the lower airways. These inflammatorychanges were absent in i.n. saline-challenged control animals that hadtreated i.p. with either saline (FIG. 8B) or OVA (not shown) with alum.

HK-X Inhibition Blocks Eosinophil Infiltration and Mucus Accumulation inAirwaves. Inflammation inhibition by HK-X markedly reduced eosinophilinflux into the lung tissue and BAL fluid of OVA sensitized/challengedmice and also prevented airway mucus release in these animals (FIGS. 8A,9A, 10A).

Eosinophil Infiltration. By morphometric analysis, the eosinophil influxinto the lung interstitium was reduced 90% by HK-X treatment (p<0.006compared to OVA without HK-X) (FIG. 11). The SLO Zileuton decreased thenumber of eosinophils in the BAL fluid by 82% (p<0.004 compared to OVAZileuton (FIG. 11). The number of eosinophils in the BAL fluid from theOVA-sensitized/challenged mice treated with the HK-X was determined as0.57+0.11×10⁵ and OVA-immunized/saline-challenged as 0.16+0.03×10⁵(control mice). Zileuton similarly decreased eosinophils recovered inthe BAL fluid of OVA-sensitized/challenged mice by 89% (p=0.0128compared to OVA without zileuton; data not shown). Vehicle controls(Trappsol™ for Zileuton studies), did not affect the lung eosinophilinfiltration observed in OVA sensitized/challenged mice.

Mucus Accumulation. Cross-sections of the upper and lower lobes of theleft lung of OVA-treated and control mice were examined by lightmicroscopy for mucus accumulation in the airways. By morphometricanalysis, 68% of the airways of control mice treated with saline had noevidence of airway mucus release, and the remainder had only a smallmucus layer observed. In contrast, OVA-immunized/challenged mice hadmorphologic evidence for widespread mucus plugging of the airways. Themajority (74%) of the airways of the OVA-treated mice had at least 30%occlusion of the airway lumen by mucus; 22% of the airways of these micehad 80% or greater mucus occlusion. When the amount of mucusglycoprotein recovered in the BAL fluid was quantitated (FIG. 12),seven-fold increase in airway mucin was demonstrated in OVA-treated micecompared to control mice (p=0.00001 OVA versus saline). HK-X treatmentblocked the airway mucus release in the OVA-treated mice (FIGS. 8A, 9A,10A).

FIGS. 8A-8C show the histopathology of the lung tissue and illustratesthe effectiveness of the treatment of the OVA induced mouse asthma bythe small peptide (HK-X) (n=8) at each group of animals. FIG. 8A is apicture of a HK-X treated mouse lung, which shows the normalcharacteristics of the features of airway (AW) and blood vessel (BV).There is very little number of cells located in the periphery of theairway tissues (arrowheads)(H & E stain, X12). FIG. 8B is a picture of amouse that received only saline injections used as a normal control. Theairway and the blood vessel are normal appearances. A few cells are seenin the airway tissue (arrowheads) (H & E stain, X120). FIG. 8C is apicture of an OVA immunized animal, which shows a profound affect bycharacteristics of eosinophil and T-cell, monocyte, and macrophageinfiltration in the airway tissue (arrowheads). The airway is plugged bythe mucus and cells (arrows) (H & E stain, X120).

FIGS. 9A-9D show the histopathology of lung tissues treated with smallpeptide (HKX) after induced with asthma in mice. This group of data areobtained from another group of mice. Histopathological evidence of theeffectiveness of the HK-X compound in treatment of asthma in mouse modelis illustrated. The HK-X compound prevents the airway mucus secretion byOVA challenge daily for a total of three days. Also the HK-X reduces thecellular infiltration during the episode of asthmatic attack. The micewere immunized with OVA intravenously and intranasally at day 1 and day14. In day 25, 26, and 27 the mice were challenged with OVA or 30minutes prior to challenge mice were received 10 μg HK-X intranasally.

FIG. 9A is a micrograph of a medium-sized airway (AW) and isrepresentative of a typical HK-X treated mouse lung. The HK-X treatedlung shows very little pathological condition as seen in the OVA treatedanimals. The airway is very clear with very little or no mucus observedin the lumen or on the epithelial cell surface. Very little amount ofcellular infiltration in the parenchyma of the airway is seen. Thesmooth muscle cell layer is uniform in thickness. (H&E stain, X150).FIG. 9B is a picture of an OVA immunized and challenged mouse lung,which shows the typical characteristics of asthma in human: a partialplugged airway (AW) lumen (arrows), a predominant feature of cellularinfiltration in the interstitium of airways and blood vessels, and aperiphery edema seen in association with blood vessels (arrowheads). (H& E stain, X75). FIG. 9C is a picture at a higher magnification of anairway of asthmatic mouse lung, which illustrates a plugged lumen.Numerous leukocyte cells are located in the basal region of the airwayepithelial cell layer (arrowheads). There are many eosinophils are seenin the parenchyma, and the smooth muscle cell layer is uneven inthickness. (H & E stain, X150). FIG. 9D is a picture of a partiallongitudinally sectioned airway of an asthmatic mouse lung, which showsthe extensive blocking of the airway lumen by released mucus (arrows).The cellular infiltration is very closely located at the area of theepithelial cell layer (arrowheads). The smooth muscle cell layer isdistracted by the infiltrating leukocyte cells and a small granuloma isoften formed. (H & E stain, X150).

FIGS. 10A-10D illustrate the histopathological data obtained fromanother group of mice (n=9). Further histopathological and histochemicalevidence is seen that the HK-X compound prevents the mucus cellinduction by OVA immunization and challenge and reduces the mucussecretion in airways. During asthmatic attack, there is an increasemucus secretion and airway constriction. The production of mucus isevident by the developing of mucus cell in the airways. Using alcianblue stains the sulfated glucosamine glucans to express themucosubstances. FIG. 10A is a picture of a HK-X treated asthmatic mouselung, which shows the airway (AW) lumen is empty and no extracellularsubstance occurred in the lumen. An adjacent blood vessel (BV) is alsopresent. The normal appearance shows no cellular infiltration or edemafluid. (H&E stain, X150). FIG. 10B is a sequential section of the sameairway as seen in FIG. 10A, which is stained with alcian blue at pH 2.4to localize the muco-substances in epithelial cells. Only a few positivecells are seen in the lumen (arrowheads). A sporadic thin-layer of bluepositive substances is evident. (Alcian blue and neutral red stain,X150). FIG. 10C is a picture of an OVA immunized and challenged mouselung, which shows a constricted airway and mucus secretion in the lumen(arrowheads). Numerous leukocytes are observed in the lung tissue. Manyof them are eosinophils. (H & E stain, X150). FIG. 10D is a similarsection as shown in FIG. 10C, which is stained with Alcian blue toindicate the muco-substances in the constricted airway. A thick-layer ofmucus is seen closely attached to the epithelial surface (arrowheads).There, more blue positive cells are seen, indicating a much higherproportion of mucus cells appeared in the airway lumen. Note alongitudinal small airway filled with mucus also is seen in thissection. (Alcian Blue & Neutral Red stain, X150).

FIG. 11 illustrates the distribution of inflammatory cells in the aveoliof lungs recovered from OVA-induced asthmatic mice. FIG. 12 illustratesthe airway plug score in airways of OVA-induced asthmatic mice. FIG. 13illustrates the white cell migration in airways OVA-induced asthmaticmice. FIG. 14 illustrates the total cells recovered from lung lavage ofOVA-induced asthmatic mice.

Induced Type II Collagen Arthritis Mouse Model

A mouse model is used to evaluate the effect of the compounds of thepresent invention on the histological, radiographic and clinicalappearance of induced type II collagen arthritis.

Autoimmune diseases cause significant and chronic morbidity anddisability. Arthritis in its many forms is representative of a family ofautoimmune diseases. In the clinical realm, rheumatoid arthritis (RA) isthe most common form of the severe arthrodysplastic disease. Allclinicians agree that RA is a progressive disease.

The histopathology of arthritic lesions occurring in murine CIA shareenormous similarities to that of RA in human patients. Thus, murine CIAis an accepted model to study potential therapeutic treatments of RA.

Materials and Methods

Mice: DBA/1 (2) male mice weighing 25 gms (Jackson Laboratories, BarHarbor, Me. or B&K Universal, Kent Wash.) are used for this work. Thisstrain of mouse is susceptible to CIA by the injection of heterologoustype II collagen. Bovine Collagen (BC), Complete Freund's Adjuvant (CFA)and Incomplete Freund's Adjuvant (ICFA) can be obtained from SigmaChemical. Antigen for immunization is processed in 0.1 M acetic acid andformulated with CFA or ICFA.

Induction of Arthritis

Immunization protocol: Mice are injected with 100.μg of type II collagenin CFA at predetermined intervals during the study period.

The mice are examined at predetermined intervals for the development ofarthritis. Presumptive evidence of arthritis includes swelling anderythema of at least one toe joint on the front and/or rear feet on twoconsecutive observations.

Confirmatory Diagnosis of Arthritis

Histological examination of joints: The toe joints of animals sacrificedat appropriate intervals are removed, fixed, decalcified, embedded, inparaffin, sectioned, and stained for observation of general cellular andstructural features and to detect cartilaginous matrix of the pannus ofeach joint, as appropriate. The degree of cellularity and areas ofinflammation are quantified by using digitization of histologicalphotomicrographs and applying standard area and point countingtechniques as described above.

Radiographic evaluation of toe joints is performed to detect theincidence of joint changes after immunization with type II collagen. Amammography imaging system has been modified for this work. The averagearea of soft tissue (pannus) of the joint is determined by analysis ofcomputer digitized radiographs, along with changes in density of theadjacent hard tissues by comparison with internal standards includedwith each radiograph. To serve as a baseline control for the changingdensity of the hard tissues and areas of panni, additional mice are usedover the same period and the density and area data compared. Thesignificance of the differences in density and area for control andexperimental mice is assessed using paired T-tests at each time point.

Arthritis Evaluation

Animals are observed daily for the onset of arthritis. An arthritisindex is derived by grading the severity of involvement of each paw on ascale from 0 to 4. Scoring is based upon the degree of peri-articularerythema and edema, as well as deformity of the joints. Swelling of hindpaws is also quantitated by measuring the thickness of the ankle fromthe medial to the lateral malledus with a constant tension caliper.

Experimental Design

To assess the anti-arthritic effect of Compound HK-X, the routes ofadministration are selected based on experience with human patientsregarding the most appropriate delivery mechanism(s).

Doses of HK-X and Prednisolone: Dosages representing divergent andputatively therapeutic levels of peptide are placed in localized sites,both by transcutaneous (TC) (absorptive) route and by injection into thefoot. Direct injection into the intraarticular space is too traumaticlikely to produce artifacts. Thus, injection of drug into the footpad(FP) adjacent to the intraarticular space is the chosen methodology.Control mice are also injected with Prednisolone (a potentanti-inflammatory documented in the treatment of experimental andclinical autoimmune diseases) as a positive control.

First, each mouse in a group of ten (plus controls) is injected withcollagen daily for 50 days. On days 3 and 18, the mouse is injected with5 or 10 .μg/kg of Compound HK-X in a solution of 0.1 M acetic acid at 1mg/ml. On day 50, the mouse is exsanguated for histologic studies.

Then, eight groups (A-I) of ten mice each are treated according to thefollowing specific protocol.

Group A is immunized with 1° CFA plus BC, 2° ICFA plus BC and notreatment is given (control).

Group B is immunized with 1° CFA plus BC, 2° ICFA plus BC andprednisolone is administered at 5 mg/kg starting on the day after 2°ICFA plus BC and continued for 20 days.

Group C is immunized with 1° CFA plus BC, 2° ICFA plus BC and CompoundHK-X is administered TC at 4 mg/kg (high dose) starting on the day after2° ICFA plus BC and continued for 20 days.

Group D is immunized with 1° CFA plus BC, 2° ICFA plus BC and CompoundHK-X is administered TC at 0.4 mg/kg (low dose) starting on the dayafter 2° ICFA plus BC and continued for 20 days.

Group E is immunized with 1° CFA plus BC, 2° ICFA plus BC and CompoundHK-X is administered TC at 4 mg/kg (high dose) starting on the day after20. ICFA plus BC and continued for 20 days.

Group F is immunized with 1° CFA plus BC, 2° ICFA plus BC and CompoundHK-X is administered TC at 0.4 mg/kg (low dose) starting on the dayafter 2° ICFA plus BC and continued for 20 days.

Group G is immunized with 1° CFA, 2° ICFA and 10 ml DMSO is administeredTC starting on the day after 2° ICFA plus BC and continued for 20 days(control).

Group H is immunized with 1° CFA, 2° ICFA and 10 ml DMSO is administeredFP starting on the day after 2° ICFA plus BC and continued for 20 days(control).

Group I is immunized with 1° CFA, 2° ICFA and 10 ml saline isadministered FP starting on the day after 2° ICFA plus BC and continuedfor 20 days (control).

Animals from each group are x-rayed immediately after 2° immunizationand immediately prior to sacrifice. Following sacrifice, feet areremoved as appropriate and processed for histological examination. Thetreatment with Compound HK-X is found to reduce the degree of arthritis.

The invention has been described in detail with reference to preferredembodiments thereof. However, it will be appreciated that, uponconsideration of the present specification and drawings, those skilledin the art may make modifications and improvements within the spirit andscope of this invention as defined by the claims.

1. A method for treating chronic inflammatory bowel disease in apatient, said method comprising administering to said patient ananti-inflammatory effective amount of a peptide or salt thereof, saidpeptide having the formula f-Met-Leu-X, wherein X is at least one memberselected from the group consisting of Tyr, Tyr-Phe (SEQ ID NO: 1),Phe-Phe (SEQ ID NO: 2) and Phe-Tyr (SEQ ID NO: 3).
 2. The method ofclaim 1, wherein an additional active ingredient is administered withsaid peptide, said active ingredient being selected from the groupconsisting of anti-leukotrienes, β₂ agonists and corticosteroids.